Depsipeptide compound

ABSTRACT

This invention relates to a novel compound which has a cytotoxic activity and TGF-β like activity for human cancer cells and is useful as an antitumor agent and to a pharmaceutical composition which contains the same.

This application is a 371 of PCT/JP00/00110, filed Jan. 12, 2000.

1. Technical Field

The present invention relates to a novel compound or a pharmaceuticallyacceptable salt thereof, which has a cytotoxic activity and TGF-β likeactivity for human cancer cells and is useful as medicaments,particularly as an antitumor agent, and to a pharmaceutical compositionwhich contains said compound as the active ingredient.

2. Background Art

It is known that mitomycin C, bleomycin, adriamycin or the like compoundderived from a microbial metabolite shows cytotoxic activity for humancancer cells, and these compounds are conventionally used as antitumoragents in the clinical field. Also, a depsipeptide compound is disclosedas an antitumor substance (European Patent Publication No. 352646).

Even at present, creation of antitumor agents having an unconventionalchemical structure and novel skeleton is under examination.

On the other hand, TGF-β is first drawing attention as a factor whichaccelerates proliferation of cells and also accelerates transformation,it was now revealed that TGF-β acts as a factor which inhibits growth ofvarious animal cells by the studies on its mechanism of actions (Cell,vol. 63, pp. 245-247, 1990). In addition, a large number of reports havebeen published regarding its relation to tumor cells (Br. Med. J., vol.296, pp. 1621-1624, 1988; Br. J. Cancer, vol. 61, pp. 612-617, 1990; Br.J. Cancer, vol. 69, pp. 1006-1009, 1994; J. Cell Physiol., vol. 172, pp.1-11, 1997; Growth Factors, vol. 7, pp. 207-213, 1992; J. Biol. Chem.,vol. 272, pp. 3967-3972, 1997; Nature, vol. 360, pp. 361-364, 1992).Also, it has been reported that TGF-β receptor acts as a tumorsuppressor gene in various tumors (International J. Hematology, vol. 65,pp. 97-104, 1997). In consequence, a compound exhibiting TGF-β likeactivity has a possibility of becoming therapeutic agents for diseasesrelated to said activity, such as an antitumor agent.

DISCLOSURE OF THE INVENTION

The invention aims at providing a novel compound which has a cytotoxicactivity and TGF-β like activity for human cancer cells and is useful asan antitumor agent and a medicament which contains the same.

As a result of intensive studies on natural compounds produced by manymicroorganisms, the present inventors have found a microorganism, amicroorganism of new species belonging to the genus Pseudomonas, whichhas the ability to produce a compound having excellent cytotoxicactivity and TGF-β like activity for human cancer cells. Thereafter, thepresent inventors have cultured said microorganism and succeeded inisolating a novel depsipeptide compound from said culture mixture, whichhas a completely different structure from that of the aforementionedknown depsipeptide compound (European Patent Publication No. 352646) interms of the following chemical structure which has hydroxyl group atthe 3-position, an R group (a group selected from any one of isopropylgroup, sec-butyl group or isobutyl group) at the 4-position and methylgroup at the 8-position, thereby accomplishing the invention.

Accordingly, the invention relates to (1) a depsipeptide compoundrepresented by the following general formula

(wherein R represents isopropyl group, sec-butyl group or isobutylgroup) or a pharmaceutically acceptable salt thereof. The invention alsorelates to a pharmaceutical composition which comprises theaforementioned depsipeptide compound or a pharmaceutically acceptablesalt thereof as the active ingredient, preferably said pharmaceuticalcomposition which is an antitumor agent.

The following describes the invention in detail. The depsipeptidecompound of the invention or a pharmaceutically acceptable salt thereofcan be obtained by culturing a said compound-producing bacteriumbelonging to the genus Pseudomonas in a nutrient medium and collectingaccumulated said compound from the culture mixture in the usual way. Anymicroorganism can be used as the microorganism to be used in theproduction method of said compound, with the proviso that it is amicroorganism which belongs to the genus Pseudomonas and has the abilityto produce said compound. As such a microorganism, a bacterial strainPseudomonas sp. Q71576 belonging to the genus Pseudomonas isolated froma soil sample collected at Mochizuki-cho, Kitasaku-gun, NaganoPrefecture, for example, can be cited. Bacterial properties of thisstrain are as follows.

1) Morphological Properties

This strain is a Gram-negative rod and has motility by polar flagella.Size of the cell is 0.7 to 0.9 μm×1.0 to 1.4 μm. Spore formation is notfound.

2) Cultural Properties

It forms light brown colonies on a bouillon agar medium. The colony iscircular and its surface is smooth. By a bouillon liquid culture, itformed pellicle on the medium surface and the entire medium was turbid.By a bouillon gelatin stab culture, it liquefied gelatin. By a litmusmilk culture, coagulation and peptonization were not found after 1 weekof the culturing.

3) Physiological Properties

TABLE 1 Physiological properties of strain Q71576 (1) Reduction ofnitrate negative Denitrification reaction negative MR test negative VPtest negative Formation of indole negative Formation of hydrogen sulfidenegative Hydrolysis of starch negative Utilization of citric acidpositive Utilization of nitrate positive Utilization of ammonium saltpositive Formation of water-soluble fluorochrome positive Ureasenegative Oxidase positive Catalase positive Growth temperature range  3to 32° C. Optimum growth temperature 10 to 24° C. Growth pH range pH 5to 9 Optimum growth pH pH 6 to 8 Growth under anaerobic conditionnegative OF test oxidation type Arginine degradation reaction positiveGrowth in 3% NaCl-added bouillon medium positive

TABLE 2 Physiological properties of strain Q71576 (2) Acid productionfrom saccharides L-Arabinose positive D-Xylose positive D-Glucosepositive D-Mannose positive D-Fructose negative Sucrose negativeInositol negative D-Mannitol negative D-Galactose negative Maltosenegative Trehalose negative Lactose negative D-Sorbitol negativeGlycerol negative Starch negative

TABLE 3 Physiological properties of strain Q71576 (3) Assimilation ofsaccharides L-Arabinose negative D-Xylose negative D-Glucose positiveD-Mannose positive D-Fructose positive Sucrose negative Inositolpositive Rhamnose negative Raffinose negative D-Mannitol positiveD-Galactose positive Maltose negative Trehalose positive Lactosenegative D-Sorbitol positive Salicin negative Melibiose negativeGlycerol positive Starch negative Xanthine positive Chitin negative

In summarizing the above microbiological properties, this strain is aGram-negative aerobic rod and has motility. Its growth temperature rangeis from 3 to 32° C., its oxidase test, catalase test, gelatinliquefaction reaction, citric acid utilization, inorganic nitrogensource utilization and arginine degradation reaction are positive, itforms acid from L-arabinose, D-xylose, D-glucose and D-mannose and theresult of the OF test is oxidation type. On the other hand, results ofthe formation of hydrogen sulfide, formation of indole, VP test,reduction of nitrate and denitrification reaction are negative.

When the aforementioned properties were referred to Bergey's Manual ofSystematic Bacteriology (1989) and other literature, this strain wasidentified as a bacterium belonging to the genus Pseudomonas and namedPseudomonas sp. Q71576.

In this connection, this strain, named Pseudomonas sp. Q71576, has beeninternationally deposited as FERM BP-6944 (deposited on Jan. 8, 1999) inNational Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology (Higashi 1-1-3, Tsukuba-shi, IbarakiPrefecture, Japan, the postal code 305-8566). Also, since microorganismsare apt to cause artificial or spontaneous mutation, the Pseudomonas sp.Q71576 includes not only microorganisms isolated from the nature butalso those which are artificially mutagenized with ultraviolet rays, Xrays, chemical drugs and the like and their spontaneous mutants.

(Production method)

The compound of the invention is obtained by culturing a microorganismwhich belongs to the genus Pseudomonas and has the ability to producethe compound of the invention. The culturing is carried out inaccordance with a general microorganism culturing method.

The medium to be used in the culturing may be any medium which containsnutrient sources utilized by Pseudomonas sp. Q71576, and a syntheticmedium, a semi-synthetic medium or a natural medium is used. Generallyknown materials can be used as the nutrients to be added to the medium.Regarding the medium composition, D-glucose, D-mannose, D-fructose,inositol, D-mannitol, D-galactose, trehalose, xanthine, starch, glucose,dextrin, glycerol, plant oil and the like can be cited as examples ofthe carbon source. As the nitrogen source, meat extract, peptone, glutenmeal, cotton seed cake, soybean powder, peanut powder, fish meal, cornsteep liquor, dry yeast, yeast extract, ammonium chloride, ammoniumsulfate, ammonium nitrate, uric acid and other organic and inorganicnitrogen sources are used. Also, sulfate, nitrate, carbonate, phosphateand the like of sodium, potassium, magnesium, calcium, zinc, iron,cobalt and the like are added as metal salts as occasion demands. Inaddition, as occasion demands, methionine, cysteine, cystine,thiosulfate, methyl oleate, lard oil, silicon oil, surface active agentand the like formation accelerating compounds or antifoaming agents canalso be added.

Regarding the culture conditions, culturing under an aerobic conditionis generally advantageous, and the culturing is carried out at atemperature of from 3 to 32° C. (cf. the aforementioned description onthe physiological properties), preferably from 20 to 25° C. Good resultsare obtained when the medium pH is adjusted to a range of approximatelyfrom 4.5 to 9, preferably from about 5 to 7.5. The culturing period isoptionally decided in response to the medium composition and temperaturecondition, but is generally from about 1 to 7 days, preferably fromabout 2 to 4 days.

In order to isolate the compound of interest of the invention from theculture mixture, techniques usually used for the extraction andpurification of metabolites produced by microorganisms can be optionallyemployed. For example, said compound among compounds in the culturemixture is extracted by adding ethyl acetate or the like organic solventwhich does not mix with water directly to the culture broth or to aculture filtrate obtained by centrifugation or by filtration afteradding a filter aid to the culture mixture. Said compound can also beextracted by allowing the culture broth to contact with an appropriatecarrier, thereby effecting adsorption of the produced compound in theculture broth to the carrier, and then eluting the compound with anappropriate solvent. For example, said compound is adsorbed by allowingit to contact with a porous adsorption resin such as Amberlite XAD-2,Diaion HP-20, Diaion CHP-20 or Diaion SP-900. Next, said compound iseluted using an organic solvent such as methanol, ethanol, acetone,butanol, acetonitrile or chloroform, alone or as a mixture, or a mixedsolution of said solvent with water. In the latter case, a fractioncontaining more higher ratio of said compound can be obtained byincreasing mixing ratio of the organic solvent from a low concentrationto a high concentration stepwise or continuously. When extracted withethyl acetate, chloroform and the like organic solvents, said compoundis extracted by adding these solvents to the culture filtrate andthoroughly shaking the mixture. Thereafter, said compound contained inthe fraction thus obtained using the above each technique can beseparated and purified with more higher purity by employing a usuallyused method such as a column chromatography which uses silica gel, ODSor the like, a centrifugal liquid-liquid partition chromatography or ahigh performance liquid chromatography (HPLC) which uses ODS.

On the other hand, it can also be separated and purified using the TGF-βlike activity as the marker, by certain means which are used in theproduction of general physiologically active compounds making use, forexample, of the difference in its solubilizing ability or solubility inappropriate solvents. As occasion demands, these methods can be usedalone or repeatedly by combining them in an optional order.

The pharmaceutically acceptable salt of the depsipeptide compound of theinvention is a salt with an inorganic or organic base, and apharmaceutically acceptable salt is desirable. Illustrative examples ofthese salts include salts with inorganic bases such as sodium,potassium, magnesium, calcium and aluminum, organic bases such asmethylamine, ethylamine and ethanolamine, and basic amino acids such aslysine and ornithine.

Also, since the compound of the invention has asymmetric carbon atomsand double bonds, stereoisomers (racemic bodies, optical isomers,diastereomers and the like) and geometrical isomers (cis-forms ortrans-forms) are present based on this. Consequently, mixtures orisolated products of these stereoisomers or geometrical isomers areincluded in the compound of the invention.

In addition, hydrates or various solvates of said compound andpolymorphism of said compound are also included in the invention.

The formulation method and administration method of the compound of theinvention are described in detail in the following.

The pharmaceutical composition which contains one or two or more of thedepsipeptide compounds of the invention or pharmaceutically acceptablesalts thereof as the active ingredient is prepared into tablets,powders, fine subtilaes, granules, capsules, pills, solutions,injections, suppositories, ointments, adhesive preparations and the likeusing generally used pharmaceutical carriers, fillers and otheradditives and administered orally or parenterally.

Clinical dose of the compound of the invention in human is optionallydecided by taking into consideration symptoms, weight, age, sex and thelike of each patient to be treated.

The solid composition for use in the oral administration according tothe invention is used in the form of tablets, powders, granules and thelike. In such a solid composition, one or more active compounds aremixed with at least one inert diluent such as lactose, mannitol,glucose, hydroxypropylcellulose, microcrystalline cellulose, starch,polyvinyl pyrrolidone or aluminum magnesium silicate. In accordance withthe usual way, the composition may contain other additives than theinert diluent, such as magnesium stearate or the like lubricant, calciumcellulose glycolate or the like disintegrating agent, lactose or thelike stabilizing agent and a solubilization assisting agent. Ifnecessary, tablets or pills may be coated with a sugar such as sucrose,gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalateor the like or with a film of a gastric or enteric compound.

The liquid composition for oral administration includes pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, elixirs and thelike and contains a generally used inert diluent such as purified wateror ethyl alcohol. In addition to the inert diluent, this composition mayalso contain a solubilization assisting agent, a moistening agent, asuspending agent and the like auxiliary agents, as well as sweeteners,flavors, aromatics and antiseptics.

The injections for parenteral administration include aseptic aqueous ornon-aqueous solutions, suspensions and emulsions. Examples of thediluent for use in the aqueous solutions and suspensions includedistilled water for injection use and physiological saline. Examples ofthe diluent for use in the non-aqueous solutions and suspensions includepropylene glycol, polyethylene glycol, olive oil or the like plant oil,ethyl alcohol or the like alcohol, polysorbate 80 (trade name) and thelike. Such a composition may further contain additive agents such as atonicity agent, an antiseptic, a moistening agent, an emulsifying agent,a dispersing agent, a stabilizing agent (e.g., lactose) and asolubilization assisting agent. These compositions are sterilized byfiltration through a bacteria retaining filter, blending of a germicideor irradiation. Alternatively, they may be used by firstly making intosterile solid compositions and then dissolving them in sterile water ora sterile solvent for injection use prior to their use.

If solubility of the compound of the invention is low, itssolubilization treatment may be carried out. The solubilizationtreatment can be effected by a known method which can be applied topharmaceutical preparations, such as a method in which surface activeagents (polyoxyethylene hydrogenated castor oil, polyoxyethylenesorbitan higher fatty acid esters, polyoxyethylene polyoxypropyleneglycols, sucrose fatty acid esters and the like) are added or a methodin which a solid dispersion of the drug is formed with a solubilizingagent such as a polymer (hydroxypropylmethylcellulose (HPMC), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) or the like water-solublepolymer or carboxymethylethylcellulose (CMEC),hydroxypropylmethylcellulose phthalate (HPMCP), a methylmethacrylate-methacrylic acid copolymer (Eudragit L, S, trade name; mfd.by Rohm & Haas) or the like enteric polymer). If necessary, a method toform a soluble salt or a method to form an inclusion compound usingcyclodextrin or the like can also be employed. The means for effectingsolubilization can be optionally changed in response to the drug ofinterest [cf., “Recent Formulation Techniques and their Application I”,I. Utsumi et al., Medical Journal 157-159 (1983) and “Pharmacy MonographNo. 1, Bioavailability”, K. Nagai et al., Soft Science, 78-82 (1988)].

BEST MODE FOR CARRYING OUT THE INVENTION

The following describes the invention illustratively with reference toexamples, but the invention is not limited to them.

EXAMPLE 1

A medium containing 10 g of glucose, 20 g of potato starch, 5 g ofpolypeptone, 5 g of yeast extract, 4 g of calcium carbonate and 1 literof distilled water (pH 7.0) was dispensed in 100 ml portions into 500 mlErlenmeyer flasks and sterilized at 120° C. for 20 minutes. Cells ofPseudomonas sp. Q71576 well grown on Bennett agar medium were scratchedoff, inoculated into the medium and cultured at 28° C. for 3 days on ashaker under a condition of 200 rotation/min to be used as the seedculture broth. Next, a medium containing 30 g of glycerol, 1 g ofglucose, 5 g of polypeptone, 5 g of meat extract, 5 g of NaCl, 0.5 g ofan antifoaming agent (NKL 5430) and 1 liter of distilled water (pH 7.0)was dispensed in 100 ml portions into 500 ml Erlenmeyer flasks andsterilized at 120° C. for 20 minutes. The aforementioned seed culturebroth was inoculated in 2 ml portions into this medium and cultured at28° C. for 3 days on a shaker under a condition of 200 rotation/min.

A 2.5 liter portion of the thus cultured broth was centrifuged at 6,000rpm for 10 minutes. The supernatant was extracted with ethyl acetate,dehydrated by adding sodium sulfate and then concentrated to drynessunder a reduced pressure. The oily crude extract was applied to a silicagel column chromatography (30 i.d.×200 mm), washed withchloroform-methanol (20:1) and then eluted with chloroform-methanol(5:1), and the active fraction was concentrated. Next, this was appliedto a Sephadex LH-20 column chromatography (20 i.d.×500 mm) to carry outgel filtration with chloroform-methanol (1:1). The active fraction wasconcentrated and then applied to a CPC (centrifugal partitionchromatography), and impurities were removed with achloroform-methanol-water (5:6:4) solvent system using an ascendingmethod. After finally concentrating the active fraction to dryness, thiswas dissolved in methanol to carry out a reverse phase HPLC (flow rate,10 ml/min) with 35% acetonitrile aqueous solution using a PEGASIL ODScolumn (20 i.d.×250 mm) manufactured by Senshu Scientific. As a result,peak of the compound A was observed at 10.8 minutes, and that of thecompound B at 15.4 minutes, and 10 mg of white powder of each of thecompounds A and B was obtained by fractionating respective peaks.

EXAMPLE 2

A medium containing 10 g of glucose, 20 g of potato starch, 5 g ofpolypeptone, 5 g of yeast extract, 4 g of calcium carbonate and 1 literof distilled water (pH 7.0) was dispensed in 100 ml portions into 500 mlErlenmeyer flasks and sterilized at 120° C. for 20 minutes. Cells ofPseudomonas sp. Q71576 well grown on Bennett agar medium were scratchedoff, inoculated into the medium and cultured at 28° C. for 3 days on ashaker under a condition of 200 rotation/min. The same medium wasdispensed in 400 ml portions into 2 liter Erlenmeyer flasks andsterilized at 120° C. for 20 minutes, and then 8 ml of theaforementioned seed culture broth was inoculated into this medium andcultured at 28° C. for 3 days on a shaker under a condition of 200rotation/min to be used as the seed culture broth. Next, a mediumcontaining 30 g of mannitol, 5 g of polypeptone, 5 g of meat extract, 5g of sodium chloride and 1 liter of tap water (pH 7.0) was dispensed in18 liter portions into three 30 liter capacity jar fermentors andsterilized at 120° C. for 20 minutes. The above seed culture broth wasinoculated in 360 ml portions into this medium and cultured at 24° C.for 64 hours under conditions of 150 rotation/min and 1 vvm.

A 50 liter portion of the culture broth separated from the cells bySharples was applied to a column packed with HP-20, washed with water,20% acetone aqueous solution and 40% methanol aqueous solution and theneluted with 80% acetone aqueous solution. An aqueous solution obtainedby concentrating the eluted fraction was extracted with chloroform andethyl acetate, and the extracts were combined, concentrated and thenapplied to a column packed with silica gel. This was eluted withchloroform-methanol (50:1), (20:1) and (10:1), and predeterminedportions of the chloroform-methanol (20:1) and (10:1) elution fractionswere combined, concentrated and then dissolved in ethanol to carry outrecrystallization, thereby obtaining 776 mg of white powder as a mixturecontaining the compounds A, B and C. By carrying out fractionation ofthe compound C elution fraction from the thus obtained powder using anODS-HPLC column (cosmosil AR-II, 20 i.d.×250 mm), 20 mg of the compoundC was obtained as white powder. Physicochemical properties of thecompounds of the invention

The compounds A, B and C which have been extracted, purified andisolated by the above techniques showed the following physicochemicalproperties.

TABLE 4 Physicochemical properties of compounds A, B and C Compound ACompound B Compound C Color and White powder White powder White powdershape Melting 135-138° C. 132-135° C. N.T. point Angle of −63.6° −58.6°−60.0° rotation (c 0.14, MeOH) (c 0.11, MeOH) (c 0.10, [α]_(D) ²⁵ MeOH)Molecular C₂₀H₃₁N₃O₆S₂ C₂₁H₃₃N₃₃O₆S₂ C₂₁H₃₃N₃O₆S₂ formula Highresolution FAB mass spectrum Found 474.1735 (M + H)⁺ 488.1865 (M + H)⁺488.1889 Calcd 474.1733 488.1889 (M + H)⁺ 488.1889 UV and End absorptionEnd absorption End absorption visible region absorption spectrum λ_(max)^(MeOH) nm (ε) IR 3400, 3350, 3400, 3350, 3400, 3320, absorption 1720,1660, 1720, 1660, 1730, 1660, spectrum 1520, 1260, 1520, 1260, 1550,1280, V_(max cm) ⁻¹  980  980  980 (KBr method) (KBr method)(reflectance method) N.T.: not tested

The ¹H and ¹³C NMR chemical shift values (in CDCl₃) of compounds A, Band C are respectively shown below.

TABLE 5 The ¹H and ¹³C NMR chemical shift values (in CDCl₃) of compoundA No. δ_(c) δ_(H) 1 171.3 2 52.2 4.21 (dq, J = 4.0, 7.5 Hz) 3 16.5 1.48(d, J = 7.5 Hz) NH 6.28 (m) 1′ 169.1 2′ 54.9 4.84 (dt, J = 3.5, 9.0 Hz)3′ 40.9 3.13 (m), 3.28 (m) NH 6.79 (d, J = 9.0 Hz) 1″ 171.7 2″ 39.5 2.68(d, J = 4.0 Hz) 3″ 69.1 4.52 (m) 4″ 63.4 2.77 (m) 5″ 29.7 2.34 (m) 6″19.7 0.90 (d, J = 7.0 Hz) 7″ 20.6 1.00 (d, J = 7.0 Hz) NH 7.38 (d, J =7.0 Hz) OH 3.09 (d, J = 10.0 Hz) 1′″ 170.8 2′″ 40.3 2.59 (d, J = 13.0Hz), 3.31 (dd, J = 7.0, 13.0 Hz) 3′″ 70.7 5.48 (m) 4′″ 128.9 5.68 (d, J= 15.0 Hz) 5′″ 133.3 6.31 (m) 6′″ 33.1 2.43 (m), 2.68 (m) 7′″ 40.9 2.73(m), 3.24 (m) The number (No.) in the table indicates respectivepositions of carbon atoms in the chemical structural formula of compoundA shown in the following.

TABLE 6 The ¹H and ¹³C NMR chemical shift values (in CDCl₃) of compoundB No. δ_(c) δ_(H) 1 171.2 2 52.2 4.22 (dq, J = 4.0, 7.0 Hz) 3 16.6 1.48(d, J = 7.0 Hz) NH 6.18 (m) 1′ 169.2 2′ 54.5 4.87 (dt, J = 3.0, 9.0 Hz)3′ 41.3 3.10 (m), 3.33 (m) NH 6.75 (d, J = 9.0 Hz) 1″ 171.8 2″ 39.5 2.70(d, J = 4.0 Hz) 3″ 68.2 4.60 (m) 4″ 61.7 2.94 (m) 5″ 36.3 2.05 (m) 6″27.1 1.21 (m), 1.53 (m) 7″ 11.5 0.89 (t, J = 7.5 Hz) 8″ 15.4 0.90 (d, J= 7.0 Hz) NH 7.25 (d, J = 7.0 Hz) OH 2.93 (m) 1′″ 170.6 2′″ 40.7 2.58(d, J = 13.0 Hz), 3.31 (dd, J = 7.0, 13.0 Hz) 3′″ 70.6 5.48 (m) 4′″128.6 5.67 (d, J = 15.0 Hz) 5′″ 133.4 6.36 (m) 6′″ 33.3 2.44 (m), 2.71(m) 7′″ 40.5 2.72 (m), 3.20 (m) The number (No.) in the table indicatesrespective positions of carbon atoms in the chemical structural formulaof compound B shown in the following.

TABLE 7 The ¹H and ¹³C NMR chemical shift values (in CDCl₃) of compoundC No. δ_(c) δ_(H) 1 171.3 2 52.3 4.22 (dq, J = 7.3, 3.7 Hz) 3 16.5 1.50(d, J = 7.3 Hz) NH 6.40 (br) 1′ 168.9 2′ 55.1 4.81 (m) 3′ 41.1 3.20 (m)NH 6.82 (d, J = 9.1 Hz) 1″ 171.4 2″ 38.8 2.68 (m) 3″ 70.7 4.35 (m) 4″56.0 3.08 (m) 5″ 38.8 1.51 (m), 2.06 (m) 6″ 25.2 1.62 (m) 7″ 21.3 0.91(d, J = 6.7 Hz) 8″ 23.4 0.91 (d, J = 6.7 Hz) NH 7.49 (d, J = 6.7 Hz) OH2.96 (br) 1′″ 170.9 2′″ 40.3 2.62 (d, J = 12.8 Hz), 3.36 (d, J = 12.8,7.3 Hz) 3′″ 70.7 5.48 (m) 4′″ 129.0 5.72 (d, J = 15.8 Hz) 5′″ 133.2 6.29(m) 6′″ 32.7 2.43 (m), 2.72 (m) 7′″ 40.5 2.74 (m), 3.31 (m) The number(No.) in the table indicates respective positions of carbon atoms in thechemical structural formula of compound C shown in the following.

Based on the above physicochemical properties, chemical structuralformulae of the compounds A, B and C were determined as follows.

Industrial Applicability

Since the compound of the invention has a cytotoxic activity and TGF-βlike activities for human cancer cells, it is useful as an antitumoragent, for example as a drug or the like for colorectal cancer, lungcancer, prostatic cancer, cervical cancer or the like.

The cytotoxic activity and TGF-β like activity of the compound of theinvention for human cancer cells were confirmed by the followingmethods.

Measurements of Cytotoxic Activity for Human Cancer Cells (1)

HeLa S3 cells adjusted to a density of 6×10⁴ cells/ml were dispensed in200 μl portions, and variations concentrations of the compound A andcompound B respectively in 4 μl portions, into a 96 well test plate, andcultured at 37° C. for 3 days in a CO₂ incubator. After the culturing,the degree of cell growth was measured using Cell Counting Kit (mfd. byDOJINDO), and the growth inhibition ration at each concentration wasobtained to calculate the IC₅₀ value. As a result, the compound A andcompound B showed cytotoxic activity for HeLa S3 cells with the IC₅₀values of 1.6 μM and 1.2 μM, respectively.

Measurement of Cytotoxic Activity for Human Cancer Cells (2)

Human colon cancer WiDr cells adjusted to a density of 6×10⁴ cells/ml,human non-small cell lung carcinoma A549 cells adjusted to a density of4×10⁴ cells/ml or 6×10⁴ cells/ml were dispensed into a 96 well testplate and cultured at 37° C. in a CO₂ incubator. After 24 hours variedconcentration of the compound A, compound B or compound C was dispensedin 100 μl portions, and the cells were cultured at 37° C. for additional72 hours in a CO₂ incubator. After the culturing, the number of cellswas determined using Sulforhodamine B, and the IC₅₀ value of eachcompound for cell growth was calculated. As a result, the compounds A, Band C showed excellent cell growth inhibition activity for respectivecell species WiDr cells, A549 cells or DU-145 cells. Though it variesdepending on the cell species, for example for A549 cells, the compoundsshowed the activity with an IC₅₀ value of 5.0 nM or less.

Measurement of TGF-β Like Activity

In order to detect the TGF-β like activity, a screening system wasconstructed making use of the expression of a reporter gene. Aluciferase gene was transfected into downstream of the PAI-1 promotergene of mink lung epithelial cells (Mv1Lu) in which expression ofplasminogen activator-1 (PAI-1) is induced by TGF-β due to excessexpression of TGF-β receptor (Journal of Biological Chemistry, vol. 262,pp. 17467-17474, 1987), (Analytical Biochemistry, vol. 216, pp. 276-284,1994).

Using the above cells, the luminous intensity obtained by adding eachcompound was used as the index of TGF-β like activity. As a result,TGF-β increased the luminous intensity induced by the expression ofPAI-1 promoter gene. In the same manner, compound A increased theluminous intensity at a concentration of from 26 nM to 100 μM, andcompound B at a concentration of from 12 nM to 100 μM.

What is claimed is:
 1. An isolated depsipeptide compound represented bythe following formula

or a pharmaceutically acceptable salt thereof, wherein R is isopropyl,sec-butyl or isobutyl group.
 2. A pharmaceutical composition whichcomprises the depsipeptide compound or a pharmaceutically acceptablesalt thereof described in claim 1 as the active ingredient.
 3. Thepharmaceutical composition according to claim 2, wherein the activeingredient is an antitumor agent.